Reovirus is a double-stranded RNA virus with a segmented genome. The receptor for the mammalian reovirus, sialic acid, is a ubiquitous molecule, therefore reovirus is capable of binding to a multitude of cells. However, most cells are not susceptible to reovirus infection and binding of reovirus to its cellular receptor results in no viral replication or virus particle production. This is probably the reason why reovirus is not known to be associated with any particular disease.
It was discovered recently that cells transformed with the ras oncogene become susceptible to reovirus infection, while their untransformed counterparts are not (Strong et al., 1998). For example, when reovirus-resistant NIH 3T3 cells were transformed with activated Ras or Sos, a protein which activates Ras, reovirus infection was enhanced. Similarly, mouse fibroblasts that are resistant to reovirus infection became susceptible after transfection with the EGF receptor gene or the v-erbB oncogene, both of which activate the ras pathway (Strong et al., 1993; Strong et al., 1996). Thus, reovirus can selectively infect and replicate in cells with an activated Ras pathway.
The ras oncogene accounts for a large percentage of mammalian tumors. Activating mutations of the ras gene itself occur in about 30% of all human tumors (Bos, 1989), primarily in pancreatic (90%), sporadic colorectal (50%) and lung (40%) carcinomas, as well as myeloid leukemia (30%). Activation of factors upstream or downstream of ras in the ras pathway is also associated with tumor. For example, overexpression of HER2/Neu/ErbB2 or the epidermal growth factor (EGF) receptor is common in breast cancer (25–30%), and overexpression of platelet-derived growth factor (PDGF) receptor or EGF receptor is prevalent in gliomas and glioblastomas (40–50%). EGF receptor and PDGF receptor are both known to activate ras upon binding to their respective ligand, and v-erbB encodes a constitutively activated receptor lacking the extracellular domain.
Since a large number of human tumors are accounted for by genetic alteration of the proto-oncogene ras or a high Ras activity, reovirus therapy is a new, promising therapy for such conditions (Coffey et al., 1998). Reovirus therapy is highly selective for Ras-associated tumor cells and leaves normal cells uninfected. Consequently, a simple and cost-effective method for the production of infectious reovirus suitable for clinical administration in human beings is needed.
Because reovirus does not pose a serious threat to human health, there has not been an intensive effort to produce reovirus efficiently. The mammalian reovirus is traditionally grown in mouse L-929 fibroblasts (Nibert et al., 1996). It has also been reported to grow in Chinese hamster ovary cells and Vero cells, an African green monkey kidney cell line (Taber et al., 1976; Davis et al., 1990). In addition, a primary culture of swine kidney was used to culture a swine reovirus (Japanese Patent 63044532A, published Feb. 25, 1988). In a study aiming at mass production of the reovirus, Berry et al. conducted an investigation of the optimal methods of culturing Vero cells and the subsequent reovirus infection (Berry et al., 1999). Vero cells were grown in either Cytodex-1 or Cultispher-G microcarriers, and culture parameters such as cell density, time course of viral growth and the ratio of cells to beads in the microcarrier were varied and virus yield determined. The study showed that the yield of virus varied greatly with the culture parameters, and complicated culture conditions (e.g. cell number per beads relative to multiplicity of infection) were required to obtain reasonable yield. Therefore, there remains a need for a simple, efficient method to produce clinically useful reovirus.